Chloride secretion by cultures of pig tracheal gland cells

Abstract

Malfunction of airway submucosal glands contributes to the pathology of cystic fibrosis (CF), and cell cultures of CF human airway glands show defects in Cl(-) and water transport. Recently, a transgenic pig model of CF (the CF pig) has been developed. Accordingly, we have developed cell cultures of pig airway gland epithelium for use in investigating alterations in gland function in CF. Our cultures form tight junctions (as evidenced by high transepithelial electrical resistance) and show high levels of active anion secretion (measured as amiloride-insensitive short-circuit current). In agreement with recent results on human airway glands, neurohumoral agents that elevate intracellular Ca(2+) potently stimulated anion secretion, while elevation of cAMP was comparatively ineffective. Our cultures express lactoferrin and lysozyme (serous gland cell markers) and MUC5B (the main mucin of airway glands). They are, therefore, potentially useful in determining if CF-related alterations in anion transport result in altered secretion of serous cell antimicrobial agents or mucus.

Fig. 1.

Standard Ussing chamber protocol. Tissues were treated sequentially with amiloride (Am, 10⁻⁵ M in mucosal bath), forskolin (For, 10⁻⁵ M in serosal and mucosal baths), methacholine (Mch, 10⁻⁵ M in both baths), ATP (10⁻⁵ M in both baths), CFTR_inh-172 (inh-172, 5 × 10⁻⁵ M in mucosal bath), flufenamic acid (FFA, 10⁻⁴ M in mucosal bath), and ouabain (On, 10⁻⁴ M in serosal bath). Record is from cells grown on Transwell insert in EGF medium with an air interface.

Fig. 2.

Effects of insert on electrical properties and protein and DNA content. Values for transepithelial electrical resistance (R_te), short-circuit current (I_sc), changes in I_sc in response to forskolin (ΔF), methacholine (ΔM), and ATP (ΔA), and protein and DNA contents are expressed relative to mean values on Transwell inserts. Open columns, Cyclopore inserts; hatched columns, Millicell-HA inserts; solid columns, Millicell CM inserts. Values are means ± SE, n = 3. Mean values for Transwell inserts are given in results. Cells were grown immersed in 2% USG.

Fig. 3.

Time dependence of baseline electrical properties. Cells were grown on Transwell inserts in 2% USG. A and B: R_te with air-interface and immersion feeding, respectively. C and D: I_sc with air-interface and immersion feeding, respectively. ○, Zero values. Regression lines have been drawn through nonzero values (●).

Fig. 4.

Confocal fluorescence immunolocalization of gland cell markers: lactoferrin (A and D), lysozyme (B and E), and MUC5B (C and F). A–C: confocal sections taken 2–5 μm below the apical membrane. D–F: z stacks. Positive staining is indicated by red. Nuclei were counterstained with Yo-Pro (green). Scale bars, 50 μm (A–C) and 20 μm (D–F). Cell sheets were confluent, so absence of nuclei merely means that the apical surface was irregular and disappeared below the focal plane in certain areas.

Fig. 5.

I_sc responses of surface epithelial cell cultures. See Fig. 1 legend for explanation of abbreviations. Record is from a cell sheet grown on a Transwell insert in EGF medium with air-interface feeding.